With over 36 classes of known anti-ribosomal agents, the bacterial ribosome has been exploited for decades as a target for antibiotic drugs. The essentiality, selectivity, and conserved nature of bacterial translation continue to make the ribosome one of the most attractive targets for the discovery of antibacterial agents. The recent elucidation of the crystal structures of the 30S and 50S ribosomal subunits from Thermus thermophilus (Wimberly et al., (2000) Nature 407, 327-339) and Haloarcula matsumorii (Ban et al., (2000) Science 289, 905-920), respectively have provided an enormous amount of detailed structural information regarding RNA and protein structure, RNA-protein interactions, and ribosome assembly. In addition, these revolutionary discoveries have provided a structural basis for understanding the mechanistic processes of translation and the action of antibiotics, and may also allow for the de novo design of new antibacterial drugs that target the translation machinery.
Ribosomal protein S8 is an excellent target for the discovery of new antibacterials. In addition to being absolutely required for the proper assembly of the 30S ribosomal subunit in E. coli, the invention provide that S8 in S. pneumoniae is essential for bacterial growth. Moreover, alterations in S8-rRNA affinity can be correlated with growth defects that result from the expression of the same mutations in E. coli (Gregory and Zimmermann, (1986), Nucleic Acids Research 14, 5761-76). Ribosomal protein S8 is also a broad-spectrum target, as it is predicted to be very well conserved among bacterial species, see Table 1. Although there is a human ribosomal protein S8, it is significantly divergent and not thought to be a functional homolog of the bacterial S8. In addition, there does not appear to be any helix 21 homology in eukaryotic 18S rRNA.
Ribosomal protein S8 is a primary rRNA-binding protein that binds directly to a central region of bacterial 16S rRNA called Helix 21. Since S8 is required for the co-operative binding of all 30S proteins, it is essential for the proper assembly of the small ribosomal subunit (Held et al., (1974) J. Biol. Chem 249, 3101-3111). Indeed, mutations within the protein have been shown to result in ribosome assembly in defects in E. coli (Geyl et al. (1977) Mol Gen Genet. 29, 331-6). The binding site of S8 within Helix 21 rRNA has been extensively characterised in E. coli, and consists of two helical segments interrupted by a very highly conserved core element of irregular structure that spans nucleotides 595-598 and 640-644 of 16S rRNA. An unusual feature of this core element, which has been elucidated by NMR studies (Kalurachchi et al., (1 997) Proc. Natl Acad. Sci USA 94, 2139-2144), is the existence of a base triple element between nucleotides A595 and the A596/U644 base pair in the E. coli rRNA. The deletion of A595 severely impairs the binding of E. coli S8 (Mougel et al., (1993) Eur J. Biochem. 215, 787-792), indicating that this nucleotide is critical for RNA-protein recognition. Interestingly, this base triple is predicted by comparative phylogenetic evidence to be nearly universally-conserved in prokaryotes (Gutell (1993), Nucleic Acids Res. 21, 3051-3054).
While the S8-RNA interactions have been extensively characterized in E. coli, and the crystal structures of both B. stearothermophilus and T. Thermophilus S8 have been solved, prior to this invention very little was known regarding the structure and RNA-binding activities of ribosomal proteins from a pathogenic bacterial species. This problem is solved by the instant invention that provides cloned, expressed, and purified Staphylococcus aureus ribosomal protein S8, and RNA-binding studies showing a specific interaction between S8 and S. aureus or E. coli helix 21 RNAs. Mutagenesis studies also provided herein have defined nucleotides in the core RNA element from S. aureus that are essential for recognition by S8 and suggest a conservation of the structure of helix 21 in this organism. Also provided herein is a crystal structure of the native S8 protein from S. aureus to 1.56 Å resolution, and superimposition of this structure into the 30S ribosomal subunit structure of T. thermophilus has provided regions of contact with a cognate rRNA. Characterisation of S. aureus S8, taken together with a recent published crystal structure of the 30S ribosomal subunit (Wimberly et al., (2000) Nature 407, 327-339) advances understanding of bacterial ribosome architecture and allows for rational design of broad-spectrum antibiotics that target the translational apparatus.
The instant invention further provides a crystal structure of the protein S8 from the small ribosomal subunit of Staphylococcus aureus in its native state. A preferred structure shows that S8 presents two surfaces that are suited to bind a cognate rRNA. On of them is a α-helix that binds into the major groove of the double stranded rRNA. A second surface is one in which conserved residues are located and are critical for binding to a cognate rRNA. S8 interacts with rRNA through this second surface by an interaction that can be described as “riding” over the double helix. Given the significant role of S8 in organising and binding rRNA for proper ribosomal function and therefore proper protein synthesis, targeting either interaction between S8 and rRNA for disruption with small molecules could result in an effective antimicrobial.